Cell Culture

a cell culture and cell technology, applied in the field of cell culture, can solve the problems of multi-stage procedures, inability to achieve empirical determination of tissue culture conditions in practice, and inability to achieve multi-stage procedures, etc., and achieve the effect of level of control

Active Publication Date: 2007-12-27
PLASTICELL LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008] The present invention provides novel cell culture techniques which are based on the perception that cell culture is better approached as a dynamic process involving serial culture steps performed in a defined sequence to achieve a desired effect. The invention recognises that sequential exposure to selected agents may be exploited to modulate cellular processes and thus achieve a level of control over these which was not previously attainable by conventional techniques.

Problems solved by technology

Owing to the cumbersome nature of conventional cell culture, empirical determination of tissue culture conditions in complex, multi-stage procedures is not feasible in practice as it involves massive work loads.

Method used

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Examples

Experimental program
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Effect test

example 1

Differentiation of ES Cell Units Using Split-Pool Cell Culture

[0191] A split-pool culture experiment was performed in order to assay tissue culture conditions that might give rise to neurons of a dopaminergic phenotype using a starter culture of undifferentiated mouse ES cells.

[0192] Undifferentiated ES cells were grown on gelatin-coated tissue culture plates in the presence of 1,400 U ml −1 of leukemia inhibitory factor (LIF;Chemicon) in ES cell medium consisting of knockout Dulbecco's minimal essential medium (DMEM; GIBCO / BRL) supplemented with 15% FCS, 100 mM MEM nonessential amino acids, 0.55 mM 2-mercaptoethanol, L-glutamine, and antibiotics (all from GIBCO / BRL). To induce embryoid body (EB) formation the cells were dissociated into a single-cell suspension by 0.05% trypsin and 0.04% EDTA in PBS and plated onto nonadherent bacterial culture dishes at a density of 2-2.5×104 cells cm−2 in the medium described above. The EBs were formed for four days and then plated onto adhesiv...

example 2

Split-Pool Cell Culture of HepG2 Cell Units

[0205] A split-pool culture experiment was performed in order to assay tissue culture conditions that might affect a particular cellular process, namely the expression and / or activity of cytochrome P450 (CyP450) metabolic enzymes. Members of this class of enzyme, such as 1A1 and 1A2, can be assayed with the use of a substrate, ethoxyresorufin, that is enzymatically hydrolyzed to produce a product, resorufin, that has distinct fluorescence characteristics that can be used to measure CyP450 enzyme activity. The expression of CyP450 enzymes can be regulated by inducer molecules, such as β-naphthoflavone, and inhibitors such as α-naphthoflavone, quinidine, or aminotriazole. It is also possible for some molecules to induce expression of a CyP450 gene(s), but inhibit activity of the enzyme product of that gene. Therefore, complicated patterns of expression and activity can arise according to the pattern of serial exposure of cells to regulatory ...

example 3

Growth of Cell Units Comprising Pluripotent Stem Cells

[0208] Pluripotent mouse ES cells expressing a Tau-GFP fusion protein were maintained on a feeder layer of mitomycin C-treated SNL cells (a STO cell derivative) in ES cell medium consisting of Iscove's medium supplemented with 15% FCS, 0.55 mM 2-mercaptoethanol, L-glutamine, antibiotics (all from GIBCO / BRL), and 1,400 U ml −1 of leukemia inhibitory factor (Chemicon) and split 1:5 every other day. ES cells and feeder cells used for the formation of cell units were transferred to a gelatin-coated flask and cultured for one day in ES medium to reduce the number of feeder cells in the culture. ES cells were trypsinised from the gelatin plates and washed with FCS-containing medium, then incubated with either Cytopore 2 or Cytodex 3 microcarriers (Amersham Biosciences) that had been hydrated in PBS, sterilised by autoclaving or 70% ethanol treatment, then washed with ES medium. The ES cells were seeded at a density of about 10 cells / m...

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Abstract

A method for determining the effect of a plurality of culture conditions on a cell, comprising the steps of: a) providing a first set of groups of cell units each comprising one or more cells, and exposing said groups to desired culture conditions; (b) pooling two or more of said groups to form at least one second pool; (c) subdividing the second pool to create a further set of groups of cell units; (d) exposing said further groups to desired culture conditions; (e) optionally, repeating steps (b)-(d) iteratively as required; and (f) optionally assessing the effect on a given cell unit of the culture conditions to which it has been exposed.

Description

FIELD OF THE INVENTION [0001] The invention relates to cell culture, and in particular to the culture of primary cells, cell lines, pluripotent cells, totipotent cells and stem cells and the regulation of their various cellular processes through modulation of cell culture conditions. The invention relates to the use of multiple culture steps under a plurality of conditions to modulate cellular pathways and provides methods for determining the effect of diverse multiple culture step regimes on cellular processes such as growth, differentiation and metabolic activity. BACKGROUND TO THE INVENTION [0002] Over recent years cell culture has become a core technology in the life sciences. Cell culture is described in ‘Basic Cell Culture’ Oxford University Press (2002) Ed. J. M. Davis; and ‘Animal Cell Culture’ Oxford University Press (2000) Ed. John R. W. Masters; both of which are incorporated herein in their entirety by reference. Cell culture provides the basis for studying cellular proc...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/06C07H19/00C12Q1/24C12Q1/68C12N5/02C12N5/00C12N5/0735
CPCC12N5/00C12N2531/00C12N2503/00C12N5/0606A61K38/00A61P43/00C12N9/93A01H4/005A01H4/008C12N1/00C12N1/20C12N5/0031C12P39/00
Inventor CHOO, YEN
Owner PLASTICELL LTD
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